Magnetic particles are used in immunological tests or a sample test. By using magnetic particles, trace molecules contained in a sample can be detected selectively and highly sensitively. Magnetic particles are formed from a magnetic material such as magnetite together with a polymeric material when necessary in a fine particle shape of about a few tens of nm to a few μm in diameter. The surface of a magnetic particle is modified by an antibody or the like to be able to specifically bind directly or indirectly to particular molecules to be detected.
For example, Jpn. Pat. Appln. KOKOKU Publication No. 63-187157 discloses a measuring method of an antigen-antibody reaction using magnetic latex. According to the measuring method of Jpn. Pat. Appln. KOKOKU Publication No. 63-187157, an antibody caused to be carried by magnetic latex and an antigen present in a liquid solvent are allowed to react in the liquid solvent. After the reaction, a magnetic field is applied to the liquid solvent to recover the magnetic latex. Next, eluent is added to the recovered magnetic latex to elute antigens having reacted with antibodies carried by the magnetic latex. Then, the magnetic latex is collected by applying a magnetic field to the eluate to separate the eluate containing eluted antigens from the magnetic latex. Then, insoluble carrier particles carrying antibodies are dispensed into the separated eluate to allow a reaction and the degree of aggregation of the reaction mixture is optically measured.
Jpn. Pat. Appln. KOKAI Publication No. 1-193647 discloses a measuring method of antigens. According to the measuring method of Jpn. Pat. Appln. KOKAI Publication No. 1-193647, insoluble carrier particles containing a magnetic substance and insoluble carrier particles containing no magnetic substance are each caused to carry antibodies. These two kinds of particles are allowed to react with antigens in a liquid. After the reaction, a magnetic field is applied to the reaction mixture to collect insoluble carrier particles containing a magnetic substance in a position of a container where light measurement is not blocked. Then, antigens are measured by detecting insoluble carrier particles containing no magnetic substance floating in the liquid based on the absorbance or scattered light.
As shown in the above examples, magnetic particles can be recovered by a magnetic force by providing a magnetic field applying means such as a magnet outside and applying a magnetic field. As a result, molecules to be detected that are bonded to magnetic particles can be separated from various kinds of impurities contained in the sample and unreacted excessive reagents. With the above action, molecules to be detected can be detected and determined selectively and highly sensitively.
Apparatuses into which magnets to apply a magnetic field or the like are incorporated have been developed as analyzers that separate molecules to be detected by using magnetic particles to detect and determine such molecules.
For example, Jpn. Pat. Appln. KOKAI Publication No. 6-213900 discloses a determination method using a magnet. According to the determination method of Jpn. Pat. Appln. KOKAI Publication No. 6-213900, a magnet is provided in a lower portion of a cuvette and a portion of specimen components is precipitated and separated by using a magnetic force generated by the provided magnet. A precipitate generated by a precipitation reagent and magnetic particles are captured at the bottom of the cuvette by the magnet and separated from a supernatant fluid. Then, the analysis of the supernatant fluid excluding the precipitate and magnetic particles is carried out.
Jpn. Pat. Appln. KOKAI Publication No. 6-160401 and Jpn. Pat. Appln. KOKAI Publication No. 7-318559 disclose an immunochemical measuring apparatus. The immunochemical measuring apparatus according to Jpn. Pat. Appln. KOKAI Publication No. 6-160401 and Jpn. Pat. Appln. KOKAI Publication No. 7-318559 is removably provided with movable magnets between cuvettes attached to the entire periphery of a rotary table. Movable magnets are inserted between cuvettes when a detection target is precipitated and separated by magnetic particles and movable magnets are removed when a detection target is not precipitated and separated.
On the other hand, WO 2008/001868 discloses a method of detecting and determining molecules to be detected by using magnetic particles. In the measuring method according to WO 2008/001868, magnetic particles are caused to selectively bind to molecules to be detected that are contained in a sample and a magnetic force is added thereto to optically measure turbidity of the mixture. Then, based on the measured turbidity, the amount of molecules to be detected is calculated. In Patent Literatures 1 and 2, magnetic particles are a means for separating antibodies and are not directly involved in optical measurement. The determination method according to WO 2008/001868 is different from the above one. That is, in the determination method according to WO 2008/001868, molecules to be detected are optically detected and thus, optical properties derived directly from magnetic particles can be measured. According to this method, special reagents such as pigments to optically detect molecules to be detected are not needed and separation and cleaning processes are simplified and therefore, the time needed for inspection is shortened.
As an example of providing a magnet that forms a magnetic field for an inspection apparatus that optically detects coloring derived from magnetic particles, WO 2008/001868 shows a configuration in which a small neodymium magnet is arranged on the side face of a cell for a spectrophotometer. Jpn. Pat. Appln. KOKAI Publication No. 2009-168636 shows an example in which a magnetic field forming means is provided in an area excluding the neighborhood of a moving path of an inspection apparatus and a removal position of an object inside a cuvette. A cleaning mechanism is arranged in the removal position. The magnetic field forming means is not provided near the removal position in order to make cleaning more efficient by preventing magnetic particles from being fixed to the side wall of a cuvette by a magnetic force. Jpn. Pat. Appln. KOKAI Publication No. 2009-168636 also shows an example in which the magnetic field forming means is arranged at predetermined intervals in a magnetic field and an example in which the magnetic field forming means is provided on the side wall of a moving path at the substantially the same height as that of an optical path.
In Patent Literatures 1 and 2, the main purpose of using magnetic particles is to separate impurities and excessive reagents contained in a test solution from molecules to be detected. In this case, a magnetic field applying means only needs to be able to provide enough magnetic field strength and an appropriate magnetic field gradient to the test solution so that the separation operation is completed within a predetermined time and many variations of the concrete structure of the magnetic field applying means and the arrangement thereof are permitted. To be concrete, as the magnetic field applying means, for example, magnets having an appropriate magnetic force are arranged close to each other on the side face or at the bottom of a cuvette to aggregate magnetic particles to be separated in a narrow range of the side wall of the container. As a result, the subsequent cleaning process to remove impurities is made more efficient. Thus, the magnet is formed in a size substantially the same as or smaller than a contact portion with the test solution of the side face of the cuvette. Similarly, in Patent Literatures 3, 4, and 5, the area of one surface of a magnet facing a cuvette is smaller than the contact surface with the test solution of the side face of the cuvette. If the purpose is to separate molecules to be detected by magnetic particles, the purpose can adequately be achieved by the above magnetic field applying means.
However, the aforementioned conventional magnetic field applying means is insufficient for the determination method as disclosed in WO 2008/001868 in which molecules to be detected are determined by optically measuring turbidity or absorbance of the test solution derived directly from magnetic particles directly or indirectly bound to molecules to be detected.
That is, when magnetic particles are injected into a sample or reagent contained in a cuvette, normally the test solution is stirred by a predetermined method immediately after the injection of magnetic particles to obtain an inspection result of excellent reproducibility by promoting a reaction between the sample and reagent. Immediately after the stirring, the concentration of magnetic particles in the test solution is spatially uniformly distributed. However, if a magnetic field is applied to the test solution after stirring by a conventional magnetic field applying means, the distribution of the magnetic field in the test solution is distorted even if a magnet is installed below the bottom of the cuvette or even if a magnet is installed on the side face of the cuvette. The concentration distribution of magnetic particles becomes more spatially non-uniform resulting from the distortion of the magnetic field distribution with the passage of time. Non-uniformity in the concentration distribution of magnetic particles leads to fluctuations of measured values of absorbance or turbidity in the determination method disclosed in WO 2008/001868.
If the concentration of magnetic particles becomes non-uniform due to a magnetic field, more specifically, problems as described below arise.
When turbidity or absorbance derived from magnetic particles is optically measured, measurement results are different in accordance with the passing location of a measuring beam in the cuvette or test solution if the concentration distribution of magnetic particles becomes non-uniform. In addition, it becomes necessary to change the inspection reagent or reaction conditions for different detection targets and thus, the fluid volume of the test solution may change from inspection item to inspection item. Non-uniformity in the concentration distribution of magnetic particles adversely affects measurement results only if the fluid volume of test solution changes even if the mixing ratio of magnetic particles is constant. Thus, each time measurement conditions or the configuration of the inspection apparatus is changed, a complicated procedure like redesigning the reagent or reaction conditions is needed to obtain appropriate measurement results. Accordingly, the cost to develop an automatic analyzer increases or the inspection time increases.
When magnetic particles and other reagents or samples are allowed to react in a magnetic field, an area in which the reaction is promoted and an area in which the reaction is less likely to occur are mixed in the test solution caused by non-uniformity in the concentration distribution of magnetic particles, leading to lower reproducibility of inspection results or an occurrence of fluctuations.
Further, while fluctuations in relative physical relationship between the test solution and the magnetic field applying means arises due to producing tolerances of the size of the cuvette or a fixing means, if a magnetic field that makes the concentration distribution of magnetic particles non-uniform is applied, inspection results vary from cuvette to cuvette due to shifting of the relative physical relationship between the test solution and the magnetic field applying means.
WO 2008/001868 shows the configuration in which a small neodymium magnet is installed on the side face of a cell for a spectrophotometer. In this example, the magnetic flux density decreases from the center toward the peripheral edge portion of the magnet with swelling lines of magnetic force to the outer side and thus, magnetic particles present in an area close to edges of the magnet in the test solution move from the outer side toward the inner side. Thus, different results of the temporal change curve of turbidity of the test solution are obtained depending on the photometry position. In addition, if the fluid volume of the test solution is different, the number of magnetic particles moving from the outer side toward the inner side changes and thus, inspection results are also considered to be affected by the fluid volume.
Jpn. Pat. Appln. KOKAI Publication No. 2009-168636 discloses an example in which a magnetic field applying means is installed under a cuvette along a moving path of the cuvette. In this case, while the magnetic flux density is high near the bottom of the test solution, the magnetic field rapidly decreases upward from the bottom of the test solution. Thus, while magnetic particles near the bottom of the test solution are quickly attracted to the bottom of the cuvette, only a weak magnetic force acts on magnetic particles in an upper portion of the test solution and the concentration of magnetic particles becomes non-uniform in an up and down direction of the cuvette. Therefore, if the photometry position or the fluid volume of the test solution changes, measurement results are affected. In addition, in Jpn. Pat. Appln. KOKAI Publication No. 2009-168636, no magnetic field applying means is provided in an area where a cleaning mechanism to remove reactants is present and the magnetic field distribution is significantly distorted in this area and thus, when the cuvette pass through this area, the concentration distribution of magnetic particles is expected to change in a complex manner, adversely affecting measurement results.
An object of an embodiment is to obtain high-precision inspection results from an automatic analyzer that determines molecules to be detected by optically measuring turbidity or absorbance of a test solution derived directly from magnetic particles.